Development of a green fermentation strategy with resource cycle for the docosahexaenoic acid production by Schizochytrium sp.

The fermentation production of docosahexaenoic acid (DHA) is an industrial process with huge consumption of freshwater resource and nutrient, such as carbon sources and nitrogen sources. In this study, seawater and fermentation wastewater were introduced into the fermentation production of DHA, which could solve the problem of fermentation industry competing with humans for freshwater. In addition, a green fermentation strategy with pH control using waste ammonia, NaOH and citric acid as well as FW recycling was proposed. It could provide a stable external environment for cell growth and lipid synthesis while alleviating the dependence on organic nitrogen sources of Schizochytrium sp. It was proved that this strategy has good industrialization potential for DHA production, and the biomass, lipid and DHA yield reached to 195.8 g/L, 74.4 g/L and 46.4 g/L in 50 L bioreactor, respectively. This study provides a green and economic bioprocess technology for DHA production by Schizochytrium sp.

Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol.

BACKGROUND: Pyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements. RESULTS: A high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room­temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter. CONCLUSION: The characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Intermittent pH control strategy in sludge anaerobic fermentation: Higher short-chain fatty acids production, lower alkali consumption, and simpler control.

The pH control to promote short-chain fatty acids (SCFAs) production during anaerobic alkaline fermentation basically focused on constant pH control. In this study, a simple and consumption-reducing intermittent pH control strategy at moderate temperature (23 ± 2 °C) was investigated with adjusting pH to 10 when naturally reduced to 8. The intermittent pH control strategy could alleviate the inhibition of acid-producing bacteria by strong alkaline and high FA concentration. Meanwhile, microbial diversity promoted by 6% and 69% while the relative abundance of acid-producing bacteria increased by 36% and 61% compared to blank and constant pH fermenters. The relative genes abundance related to amino acid metabolism and fatty acid production were mostly promoted and led to enhanced SCFAs production. In the long-term fermenter, the intermittent pH control strategy could result in a 68% reduction in alkali consumption and a 37% increase in SCFAs production compared to that of the constant pH at 10.

Identification and a phased pH control strategy of diosgenin bio-synthesized by an endogenous Bacillus licheniformis Syt1 derived from Dioscorea zingiberensis C. H. Wright.

Diosgenin is widely used as one precursor of steroidal drugs in pharmaceutical industry. Currently, there is no choice but to traditionally extract diosgenin from Dioscorea zingiberensis C. H. Wright (DZW) or other plants. In this work, an environmentally friendly approach, in which diosgenin can be bio-synthesized by the endophytic bacterium Bacillus licheniformis Syt1 isolated from DZW, is proposed. Diosgenin produced by the strain was identified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). The thermal gravimetric analysis (TGA) showed that the melting point of the diosgenin product was 204 °C. The optical rotation measurement exhibited that the optical rotation was α20589 = - 126.1° ± 1.5° (chloroform, c = 1%): negative sign means that the product is left-handed, which is very important to further produce steroid hormone drugs. Cholesterol may be the intermediate product in the diosgenin biosynthesis pathway. In the batch fermentation process to produce diosgenin using the strain, pH values played an important role. A phased pH control strategy from 5.5 to 7.5 was proved to be more effective to improve production yield than any single pH control, which could get the highest diosgenin yield of 85 ± 8.6 mg L-1. The proposed method may replace phyto-chemistry extraction to produce diosgenin in the industry in the future.Key points• An endophytic Bacillus licheniformis Syt1 derived from host can produce diosgenin.• A dynamic pH industrial control strategy is better than any single pH control.• Proposed diosgenin-produced method hopefully replaces phyto-chemistry extraction.

Meta-fermentation system with a mixed culture for the production of optically pure l-lactic acid can be reconstructed using the minimum isolates with a simplified pH control strategy.

Meta-l-lactic acid fermentation from non-treated kitchen refuse was reconstructed using a combination of isolated bacterial strains under several pH control strategies. The meta-fermentation system was successfully reconstructed using a combination of Weizmannia coagulans MN-07, Caldibacillus thermoamylovorans OM55-6, and Caldibacillus hisashii N-11 strains. Additionally, a simplified constant pH control strategy was employed, which decreased fermentation time and increased production. The optimum pH (6.5) for the reconstructed meta-fermentation was favorable for the respective pure cultures of the three selected strains. The l-lactic acid production performance of the reconstructed meta-fermentation system was as follows: concentration, 24.5 g L-1 ; optical purity, 100%; productivity, 0.341 g L-1 h-1 ; yield, 1.06 g g-1 . These results indicated that constant pH control was effective in the reconstructed meta-fermentation with the best performance of l-lactic acid production at pH optimal for the selected bacterial growth, while the switching from swing pH control would suppress the activities of unfavorable bacterial species in un-isolated meta-fermentation.

Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 µM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.

Enhanced isopropanol-butanol-ethanol mixture production through manipulation of intracellular NAD(P)H level in the recombinant Clostridium acetobutylicum XY16.

BACKGROUND: The formation of by-products, mainly acetone in acetone-butanol-ethanol (ABE) fermentation, significantly affects the solvent yield and downstream separation process. In this study, we genetically engineered Clostridium acetobutylicum XY16 isolated by our lab to eliminate acetone production and altered ABE to isopropanol-butanol-ethanol (IBE). Meanwhile, process optimization under pH control strategies and supplementation of calcium carbonate were adopted to investigate the interaction between the reducing force of the metabolic networks and IBE production. RESULTS: After successful introduction of secondary alcohol dehydrogenase into C. acetobutylicum XY16, the recombinant XY16 harboring pSADH could completely eliminate acetone production and convert it into isopropanol, indicating great potential for large-scale production of IBE mixtures. Especially, pH could significantly improve final solvent titer through regulation of NADH and NADPH levels in vivo. Under the optimal pH level of 4.8, the total IBE production was significantly increased from 3.88 to 16.09 g/L with final 9.97, 4.98 and 1.14 g/L of butanol, isopropanol, and ethanol. Meanwhile, NADH and NADPH levels were maintained at optimal levels for IBE formation compared to the control one without pH adjustment. Furthermore, calcium carbonate could play dual roles as both buffering agency and activator for NAD kinase (NADK), and supplementation of 10 g/L calcium carbonate could finally improve the IBE production to 17.77 g/L with 10.51, 6.02, and 1.24 g/L of butanol, isopropanol, and ethanol. CONCLUSION: The complete conversion of acetone into isopropanol in the recombinant C. acetobutylicum XY16 harboring pSADH could alter ABE to IBE. pH control strategies and supplementation of calcium carbonate were effective in obtaining high IBE titer with high isopropanol production. The analysis of redox cofactor perturbation indicates that the availability of NAD(P)H is the main driving force for the improvement of IBE production.

Enhanced fed-batch production of pyrroloquinoline quinine in Methylobacillus sp. CCTCC M2016079 with a two-stage pH control strategy.

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (µ x ) and specific PQQ formation rate (µ p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.

Novel pH control strategy for efficient production of optically active l-lactic acid from kitchen refuse using a mixed culture system.

Uninvestigated control factors of meta-fermentation, the fermentative production of pure chemicals and fuels in a mixed culture system, were examined for production of optically pure l-lactic acid (LA) from food waste. In meta-fermentations by pH swing control, l-LA production with 100% optical purity (OPl-LA) was achieved even using unsterilized model kitchen refuse medium with preferential proliferation of l-LA-producing Bacillus coagulans, a minor member in the seed, whereas agitation decreased OPl-LA drastically. pH constant control shortened the fermentation time but decreased OPl-LA and LA selectivity (SLA) by stimulating growth of heterofermentative Bacillus thermoamylovorans. Deliberately switching from pH swing control to constant control exhibited the best performance for l-LA production: maximum accumulation, 39.2gL(-1); OPl-LA, 100%; SLA, 96.6%; productivity, 1.09gL(-1)h(-1). These results present a novel pH control strategy for efficient l-LA production in meta-fermentation based on a concept different from that of pure culture systems.